Growth hormone is a polypeptide hormone secreted by the anterior pituitary in mammals. Dependent on species growth hormone is a protein composed of approximately 190 amino acid residues corresponding to a molecular weight of approximately 22 kDa. Growth hormone binds to and signals through cell surface receptors, the growth hormone receptors (GHR). Growth hormone plays a key role in promoting growth, maintaining normal body composition, anabolism and lipid metabolism. It also has direct effects on intermediate metabolism, such as decreased glucose uptake, increased lipolysis, increased amino acid uptake and protein synthesis. The hormone also exerts effects on other tissues including adipose tissue, liver, intestine, kidney, skeleton, connective tissue and muscle. Recombinant human growth hormone (hGH) has been produced and commercially available as, for ex: Genotropin™ (Pharmacia Upjohn), Nutropin™ and Protropin™ (Genentech), Humatrope™ (Eli Lilly), Serostim™ (Serono), Norditropin (Novo Nordisk), Omnitrope (Sandoz), Nutropin Depot (Genentech and Alkermes). Additionally, an analogue with an additional methionine residue at the N-terminal end is also marketed as, for ex: Somatonorm™ (Pharmacia Upjohn/Pfizer).
Growth hormone shares a common topology with the other members of the growth hormone family of proteins, Prolactin (PRL) and Placental Lactogen (PL). Growth hormone is classified as a four-helix bundle protein (FIG. 1) exhibiting an “up-up-down-down” topology with two conserved disulphide linkages. Specifically, wild-type human Growth hormone (hGH) is composed of 191 amino acid residues and has four cysteine residues at positions 53, 165, 182 and 189, which stabilizes the three dimensional structure of the protein by forming two intramolecular disulphide bonds connecting C53 with C165 and C182 with C189, respectively (FIG. 1). The structure of hGH has been experimentally determined by X-ray crystallography in the free form (Chantalet L. et al Protein and Peptide Letters 3, 333-340, (1995)) and in complex with its binding protein (the extra cellular domain of the human GHR (hGHR)) (Devos, A. M. et al Science 255, 306-312, (1992)). These structures have been deposited in the Protein Data Bank (PDB) and are publicly available (PDB accession codes 1HGU and 1HWG, respectively). Thus, from the published hGH structures residues important for hGH binding to hGHR can be identified. Furthermore, the dynamic properties of hGH has been studied by Nuclear Magnetic Resonance (NMR) spectroscopy (Kasimova M. R. et al. J. Mol. Biol. 318 679-695, (2002)). In combination, the X-ray and NMR data can distinguish regions of hGH which are well structured and well defined from regions which are less structured and dynamic. Less structured and dynamic regions of hGH are expected to be particularly susceptible to proteolytic cleavage and proper stabilization of such regions would lead to improved proteolytic stability.
hGH has been subject to extensive mutagenesis in attempts to produce hGH analogues with desired chemical or biological properties. Specifically, cysteine mutants for several purposes have been described.
US 2003/0162949 disclose cysteine variants of members of the GH supergene family. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue
WO 02/055532 describes genetically engineered hGH mutants having at least one non-polypeptide moiety covalently attached, particularly hGH mutants where a introduced cysteine residue was used for pegylation.
U.S. Pat. No. 5,951,972 describes physiologically active derivatized natural and recombinant mammalian and human proteins and polypeptides wherein at least one-naturally-occurring or incorporated cysteine residue within the protein is derivatized with various substituents.
The proteolytic cleavage of hGH has been studied in detail. The long loop composed of residues 128 to 154 has putative cleavage sites for several proteases, such as thrombin, plasmin, collagenase, subtilisin and chymotrypsin-like serine proteases. Accordingly, this part of hGH has been shown to be particularly susceptible to proteolytic cleavage (Lewis, U. J. Ann. Rev. Physiol. 46, 33-42, (1984)). Enzymes reported to degrade hGH include thrombin, plasmin, subtilisin, chymotrypsin-like serine proteinases and kallikreins.
The degradation of hGH in rat tissue has been investigated (Garcia-Barros et al. J. Endocrinol. Invest. 23, 748-754, (2000)).
In rat thyroid gland chymotrypsin-like proteases, favouring cleavage at bulky and lipophilic amino acid residues, were found initially to cleave the peptide bond between Y143 and S144 resulting in a two chain molecule, followed by cleavage between Y42 and S43, liberating the N-terminal peptide F1-Y42. The split loop in the two chain molecule is processed further by cleavage between F146 and D147 by chymotrypsin-like proteases and further by the action of carboxypeptidases.
Several methods to produce hGH analogues stabilized towards proteolytic degradation have been reported.
Alam et al., J. Biotech. 65, 183-190, (1998) designed hGH mutants resistant to thrombin and plasmin by specific point mutations. Thrombin cleaves hGH specifically between R134 and T135, and the double mutant R134D, T135P yielded a hGH variant resistant to cleavage by thrombin, and the triple mutant R134D, T135P, K140A resulted in resistance to plasmin. Furthermore, the latter hGH mutant was resistant to proteolysis by human plasma over a period of 7 days.
EP 534568 describes hGH mutants stabilized towards proteolytic degradation by mutating R134 to alanine, leucine, threonine, phenylalanine, proline or histidine.
WO 2004/022593/Nautilus describes general high through-put directed evolution methods to produce modified cytokines, including GH variants, with increased proteolytic stability.
WO 2006/048777/Nautilus specifically describes modified hGH analogues with improved proteolytic stability. The analogues contain one to five mutations at positions 1-55, 57, 58, 60-63, 67-87, 89-91, 93, 95-100, 102-128, 131-132, 135-139, 141, 142, 144, 148-182, 184, 185 and 187-191. Introduction of cysteine residues can potentially lead to the formation of undesired disulfide linked dimers and in WO 2006/048777 the substitution of amino acid residues by cysteine is specifically excluded from the scope; in WO 2006/048777 (p. 65) it is stated: “The replacement of amino acids by cysteine residues is explicitly avoided since this change would potentially lead to the formation of intermolecular disulfide bonds”.
There is an obvious need to develop hGH compounds which are resistant to proteolytic degradation. Such stabilized compounds should exhibit increased stability towards proteolytic cleavage while retaining the desired biological properties of hGH. Such GH molecules would have increased stability, slower clearance and/or prolong in vivo half-life.
Furthermore it is well-known to modify the properties and characteristics of peptides by conjugating groups to the peptide which duly changes the properties of the peptide. Such conjugation generally requires some functional group in the peptide to react with another functional group in a conjugating group. Typically, amino groups, such as the N-terminal amino group or the ∈-amino group in lysines, have been used in combination with a suitable acylating reagent. Alternatively, polyethylene glycol (PEG) or derivatives thereof may be attached to proteins. For a review, see Exp. Opion. Ther. Patent. 14, 859-894, (2004). It has been shown that the attachment of PEG to growth hormone may have a positive effect on the plasma half-life of growth hormone, WO 03/044056.
The use of carboxypeptidases to modify the C-terminal of peptides has been described earlier. WO 92/05271 discloses the use of carboxypeptidases and nucleophilic compounds to amidate the C-terminal carboxy group, and WO 98/38285 discloses variants of carboxypeptidase Y particular suitable for this purpose.
EP 243 929 discloses the use of carboxypeptidase to incorporate polypeptides, reporter groups or cytotoxic agents into the C-terminal of proteins or polypeptides.
WO 2005/035553 describes methods for selective conjugation of peptides by enzymatically incorporating a functional group at the C-terminal of a peptide.
Activated halogen derivatives and maleimides represent some of the most common used functional groups when incorporating conjugates to sulfhydryl groups in peptides (G. T. Hermanson in Bioconjugate Techniques 2. Ed. 2008, Elsevier).
Transglutaminase has previously been used to alter the properties of peptides. In the food industry and particular in the diary industry many techniques are available to e.g. cross-bind peptides using transglutaminases. Other documents disclose the use of transglutaminase to alter the properties of physiologically active peptides. EP 950665, EP 785276 and Sato, Adv. Drug Delivery Rev. 54, 487-504, (2002) disclose the direct reaction between peptides comprising at least one Gln and amine-functionalised PEG or similar ligands in the presence of transglutaminase, and Wada, Biotech. Lett. 23, 1367-1372, (2001) discloses the direct conjugation of β-lactoglobulin with fatty acids by means of transglutaminase. The international patent application published as WO 2005/070468 discloses the use of transglutaminase to incorporate a handle whereto conjugating groups can be attached.
Growth hormone is a key hormone involved in the regulation of not only somatic growth, but also in the regulation of metabolism of proteins, carbohydrates and lipids. The major effect of growth hormone is to promote growth. Human growth hormone is a 191 amino acid residue protein with the sequence: FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).
Administration of human growth hormone and closely related variants thereof is used to treat a variety of growth hormone deficiency related diseases. Being a polypeptide, growth hormone is administered parenterally, i.e., by means of a needle. Growth hormone is, furthermore, characterised by a relative short half-life, hence frequent administrations are required with the corresponding pain and inconvenience for the patient. Hence, there is still a need for the provision of growth hormone compounds with improved pharmacological properties, such as e.g. prolonged half-life.
The present invention provides novel growth hormone compound conjugates with improved pharmakinetic and pharmacological properties as well as methods for their production.